The invention relates in general to stable maintenance of a plasmid, and in particular to that of a plasmid containing a gene useful in gene therapy.
The stable maintenance of a plasmid, particularly at high copy number, is important for the preparation of plasmid DNA. However, extrachromosomal DNA carried in host cells is inherently unstable in cell culture because cultured cells which contain plasmids usually have an increased metabolic burden compared to plasmid-free segregant cells. In efforts to maintain plasmid stability and decrease metabolic burden, plasmids engineered to contain dominant selectable markers have been routinely used. During scale-up fermentation of bacterial or yeast host strains, the presence of the selecting agent prevents plasmid loss and overgrowth by cells not burdened by the effort of replication and maintenance of plasmid DNA.
Antibiotic resistance genes, for example encoding resistance to antibiotics such as ampicillin, kanamycin or tetracycline, are the most common dominant selectable markers used in molecular biology cloning and fermentation procedures for the production of recombinant proteins or plasmid DNA. For continuous fermentation in the presence of an antibiotic, selective pressure is lost because the antibiotic loses activity over time due to culture dilution or degradation by the host cell. Therefore, some of the more successful methods for maintaining plasmids do not utilize antibiotic selection but rather rely on a mutant host which is unable to synthesize an amino acid, inserting the gene which provides for this synthesis in the plasmid. Other solutions which prevent the takeover of a culture by plasmid-free segregant involve placing a gene coding for a toxic product in the chromosome and then including a corresponding repressor system in the plasmid. Plasmid-free cells are effectively killed upon segregation.
Even with selective pressure, however, plasmid-free cells may continue to grow due to leakage of the complementing product of the selective gene from plasmid-bearing cells, lowering the total plasmid productivity of the culture. In addition, the use of genes for antibiotic resistance or other dominant selectable markers on vectors intended for gene therapy has raised potential problems related to expression of those genes in the target mammalian cell or host mammalian organism. Promiscuous expression of plasmid-borne genes, such as drug-resistance or nutritional markers, of the host cell (e.g. a yeast or bacteria) in the target mammalian cell may lead to its destruction and/or to an antigenic response to the gene product in the mammal. There are also concerns regarding contamination of the final product with the antibiotic used for plasmid selection in culture, with the potential induction of a severe immune response to the antibiotic, e.g., anaphylactic shock. The widespread use of bacterial genes for antibiotic resistance also will ultimately result in their transfer to the bacterial population as a whole.
The stable maintenance of plasmids at high copy number in transfected cells is also of importance in ex vivo gene therapy. Following the administration of a transfected cell, such as a microorganism or other cell, to a recipient organism (e.g., a mammal) for therapy, it becomes difficult or impossible to maintain plasmid DNA within the transplanted cells because it is difficult to select for a plasmid in vivo. For example, use of the in vitro selection compound may be contraindicated in the recipient, as would be the deletion of a given biochemical constituent (such as an amino acid) from the enviroment surrounding the transplanted cells, were such a feat of biological engineering as the latter technically feasible. As is true for in vivo nucleic acid delivery methods, it is advantageous to remove from the plasmids bearing the therapeutic- or other gene of interest genes that are not relevant to the therapeutic application, in order to minimize potential risks stemming from transmission of their products to the recipient, which may provoke side effects such as a toxic or anaphylactic response.
There is, therefore, a need for a method of plasmid maintenance that does not require the presence of extraneous plasmid-borne host genes or antibiotic selection.
The invention encompasses a transformed host cell containing a plasmid comprising an operator susceptible to binding by a repressor expressed in trans, a first chromosomal gene encoding the repressor, and a second chromosomal gene that is functionally associated with an operator and essential for cell growth, wherein the plasmid is present in the cell in sufficient numbers to titrate the repressor such that the essential gene is expressed, thereby permitting cell growth.
As used herein, xe2x80x9cfunctionally associatedxe2x80x9d or xe2x80x9coperatively associatedxe2x80x9d, with respect to an operator sequence and an associated gene, means that the operator is linked in cis to the gene such that expression of the gene is susceptible to repression upon binding of a repressor to the operator. It will be understood by one of skill in the art that the operator sequence present on the plasmid need not be a sequence that is identical to the operator sequence on the chromosomal gene, in that the plasmid operator need only consist of the minimal sequences necessary for binding the repressor that represses transcription of the chromosomal gene. It will also be understood that mutated operator sequences are also useful according to the invention, for example, sequences having one or more nucleotides inserted, deleted, or substituted which result in increased or decreased affinity for the corresponding repressor. As used herein, xe2x80x9ccell growthxe2x80x9d refers to increasing numbers of cells in a culture medium over time, and also refers to cell survival where the number of cells does not increase over time, but rather the number of live cells does not decrease over time.
Preferably, the repressor gene encodes one of the lac repressor, the xcex repressor, the E. coli trp repressor, the E. coli galR repressor, and the E. coli araC repressor. As described above, each repressor is operative in trans with a trans-associated operator sequence that is present both in the chromosome and on the plasmid. The invention contemplates the presence of one or more repressor genes on the host chromosome, e.g., one, two or three repressor genes, in order to ensure plasmid stability where one chromosomal repressor gene becomes mutated or deleted.
Preferred operator sequences therefore include the lac operator, the xcex operator, the trp operator, the gal operator, and the ara operator. If desired, the corresponding promoter may be functionally associated with its operator. Note that bacterial repressor/operator systems are of use in yeast including, but not limited to, the Lac repressor/operator pair, which may be used to block access of positive regulators of yeast transcription to their respective binding sites (e.g. the binding of Gal4p to the sequence CGGN5(A/T)N5CCG [SEQ ID NO: 1].
In other preferred embodiments, the cell is a bacterial cell that may be either gram negative or positive, for example, E. coli, Listeria, Shigella, Clostridium, Salmonella, Bacillus or Lactococcus. Alternatively, the host cell may be a yeast, mycobacterial, slime mold, algal or fungal cell, or other cell of the Domain Arachaea (including archaebacteria), phylum Protista, or animal or plant kingdom.
More than one different essential chromosomal gene may be present in the cell chromosome, wherein two or more essential genes are linked to an operator and are therefore susceptible to repression by the repressor; in this way, accidental de-repression of a single essential gene (e.g. through mutation of its associated operator) cannot result in the growth of plasmid-free cells, since at least one other essential gene remains repressed. In one preferred embodiment of the invention, the gene encoding the repressor protein is present in two or three copies at different locations in the chromosome to guard against loss of repressor expression at one chromosomal location. It is also contemplated that a single essential gene may be operatively linked to more than one operator sequence, such that a mutation in any one such element will be insufficient to de-repress transcription of the gene.
Preferred essential genes that are located on the host chromosome include but are not limited to genes falling within the following categories: genes encoding products related to the biosynthesis of cell metabolites, genes whose products are involved in carbon metabolism, genes coding for antibiotic resistance, and genes encoding biosynthesis or regulation of macromolecules, e.g., genes essential for DNA and/or RNA synthesis and replication functions.
In preferred embodiments, the plasmid comprises an origin of replication permitting replication of about 10-50 copies, 40-200 or 100-200 copies of the plasmid per host cell.
Examples of such plasmids include, but are not limited to, bacterial plasmids pBR322, pUC, pHETK, pT181, pMB1, pNZ2123 and pIL253, and yeast plasmids YRp and YEp.
It is preferred that the plasmid comprises a cloning site for insertion of a gene of interest.
In one especially preferred embodiment of the invention, the plasmid further comprises a gene of interest operatively associated with control sequences for expression in a mammalian, preferably a human, cell. Examples of such genes are known in the art and disclosed herein. If desired, the gene of interest will not have a host cell promoter and therefore will not be expressed in the host cell. In addition, if desired, the gene of interest may be associated with the plasmid operator sequence such that expression of this gene is repressible upon growth of the plasmid-transformed host cell. These serve to reduce the metabolic burden to the host cell of producing the encoded protein of interest. Alternatively, if expression of the gene of interest is desired, e.g., where it is desirable to produce and isolate the encoded product, the operator need not be positioned so as to repress expression of the gene of interest upon cell growth and expression of the gene of interest may be driven by a host cell promoter.
As used herein, the terms xe2x80x9cmammalianxe2x80x9d or xe2x80x9cmammalxe2x80x9d refer to any member of the class Mammalia, including a human.
Preferably, the plasmid consists essentially of an operator susceptible to binding by a repressor, an origin of replication, and a cloning site for insertion of a gene of interest.
As used herein, xe2x80x9cconsists essentially ofxe2x80x9d means that the plasmid contains only those sequences necessary for maintaining the plasmid in the host strain, and a cloning site for insertion of a gene of interest. That is, the plasmid does not contain sequences that are unnecessary to its survival in the host (e.g., bacterial, yeast or other) strain.
As used herein, xe2x80x9corigin of replicationxe2x80x9d refers to those sequences on the plasmid that are necessary for maintaining the plasmid at a given copy number per host cell.
Preferably, the plasmid is about 1000 bp in length.
It is also preferred that the plasmid is about 2,500 bp in length.
In another preferred embodiment, the plasmid is about 5,000 bp in length.
In another aspect, the invention provides a plasmid such as that described above, e.g., consisting essentially of an operator susceptible to binding by a repressor, an origin of replication, and a cloning site for insertion of a gene of interest. It is contemplated that the DNA contained in the plasmid that is other than the operator sequence, the origin of replication, and the cloning site is non-coding DNA.
Preferably, the plasmid further comprises a said gene of interest cloned into said cloning site, particularly a gene of interest operatively associated with control sequences for expression in a mammalian, preferably a human, cell.
The minimal plasmid possesses the considerable advantage of containing only minimal bacterial DNA sequences, and thus considerably reduces the problems associated with the introduction of bacterial DNA sequences into mammalian cell lines, for example, where a plasmid is intended as a vector for gene therapy. Thus, problems that are avoided according to the invention include expression of plasmid-borne bacterial, yeast or other host genes in a mammalian target cell which lead to destruction of the target cell or the mammalian host itself, or which lead to development of an immune response to the foreign DNA or to products encoded by such sequences.
The invention also provides a method of maintaining a plasmid in a host cell, comprising the step of culturing a cell containing a plasmid comprising an operator susceptible to binding by a repressor, a first chromosomal gene encoding the repressor, and a second chromosomal gene that is functionally associated with the operator and is essential for cell growth, wherein the plasmid is present in the cell in sufficient numbers to titrate the repressor such that the essential gene is expressed, thereby permitting cell growth, for a time and under conditions sufficient to permit the cell to grow.
Another aspect of the present invention is a method of producing plasmid DNA, comprising culturing a cell comprising a first chromosomal gene encoding a repressor, a second chromosomal gene that is functionally associated with an operator susceptible to binding by the repressor and is essential for cell growth, and a plasmid comprising the operator, wherein the plasmid is present in the cell in sufficient numbers to titrate the repressor such that the essential gene is expressed, thereby permitting cell growth, for a time and under conditions sufficient to permit the cell to grow, and isolating plasmid DNA from the cell.
Preferably, the plasmid further comprises a gene encoding a recombinant protein, which gene is functionally linked to sequences which cause the gene to be expressed in a mammalian cell, e.g. a human cell.
The invention additionally encompasses a method of producing a recombinant protein, comprising culturing a cell containing comprising a first chromosomal gene encoding a repressor, a second chromosomal gene that is functionally associated with an operator susceptible to binding by the repressor and is essential for cell growth, and a plasmid comprising the operator and a gene encoding a recombinant protein, which gene is expressed in the cell, wherein the plasmid is present in the cell in sufficient numbers to titrate the repressor such that the essential gene is expressed, thereby permitting cell growth, for a time and under conditions sufficient to permit the cell to grow, and isolating the recombinant protein from the cell.
It is contemplated that the recombinant protein is a protein of therapeutic benefit to a human.
Production of a recombinant protein using the repressor titration system described herein confers a reduced metabolic burden on the host cell in that the only coding region on the plasmid is the gene encoding the recombinant protein. Therefore, the host cell need not support production of plasmid-encoded proteins other than the recombinant protein.
The repressor titration system described herein enables the stable maintenance of plasmids in moderate or high copy number without the use of plasmid-encoded dominant selectable markers, such as for antibiotic resistance, and can be used with any host that can support a trans-acting repressor/operator system. One advantage of the invention is in its reliance on plasmid maintenance other than by antibiotic selection of plasmid-bearing cells. That is, there is no loss of selective pressure during fermentation due to loss of activity of an antibiotic. The absence of dominant selectable markers, such as antibiotic resistance genes or nutritional markers, on the plasmid, as described herein, is also advantageous in that it avoids the potentially serious problems related to expression of those genes in the target mammalian cell. The invention thus also avoids contamination of a product intended for gene therapy with the antibiotic used for selection of the gene therapy vector. In addition, the invention avoids the potential induction of a severe immune response to such antibiotics, e.g., anaphylactic shock.
One considerable advantage to the non-antibiotic plasmid selection system described herein is that it avoids wide-spread use of bacterial genes encoding antibiotic resistance, which use tends to promote transfer of such genes in the bacterial population as a whole.
Another aspect of the present invention is a method of delivering a stably-maintained plasmid to a recipient organism, comprising culturing a cell comprising a first chromosomal gene encoding a repressor, a second chromosomal gene that is functionally associated with an operator susceptible to binding by the repressor and is essential for cell growth, and a plasmid comprising the operator, wherein the plasmid is present in the cell in sufficient numbers to titrate the repressor such that the essential gene is expressed, thereby permitting cell growth, for a time and under conditions sufficient to permit the cell to grow, and transplanting the cell to the recipient organism under conditions sufficient to permit the cell containing the plasmid to be present in the recipient organism after being so transplanted.
As used herein, the term xe2x80x9corganismxe2x80x9d refers to all multicellular life-forms; such organisms may provide cells which may serve as host cells for a plasmid of the invention or may serve as recipients of a transformed host cell of the invention. Organisms that are of particular use as host-cell recipients are mammals.
In contrast, the term xe2x80x9cmicroorganismxe2x80x9d refers to any unicellular organism. Microorganisms are of use in the invention as plasmid host cells, and include (but are not limited to) yeast, bacteria, members of the Domain Arachaea and the phylum Protista, slime molds, unicellular fungi and algae
Preferably, the cell is a yeast cell.
In another preferred embodiment, the cell is a bacterial cell.
It is preferred that the transplanting comprises injection of the cell into the recipient organism.
The invention also provides a method of maintaining a host cell containing an exogenous DNA in a host organism, comprising the steps of culturing a cell comprising a first chromosomal gene encoding a repressor, a second chromosomal gene that is functionally associated with an operator susceptible to binding by the repressor and is essential for cell growth, and a plasmid comprising the operator and a gene of interest which is expressed in the cell, wherein the plasmid is present in the cell in sufficient numbers to titrate the repressor such that the essential gene is expressed, thereby permitting cell growth, for a time and under conditions sufficient to permit the cell to grow, and transplanting the cell to the recipient organism under conditions sufficient to permit the cell to produce a product encoded by the gene of interest in the organism, wherein the cell containing the plasmid is present in the organism after being so transplanted. A final aspect of the present invention is a pharmaceutical composition comprising a cell comprising a first chromosomal gene encoding a repressor, a second chromosomal gene that is functionally associated with an operator susceptible to binding by the repressor and is essential for cell growth, and a plasmid comprising the operator and a therapeutic gene which is expressed in the cell, wherein the plasmid is present in the cell in sufficient numbers to titrate the repressor such that the essential gene is expressed, thereby permitting cell growth, and a pharmaceutically-acceptable carrier.
Pharmaceutically-acceptable carriers of use in the invention include, but are not limited to, aqueous solutions, such as physiological buffers (e.g. saline), cell culture media, or other excipients or adjuvants as are known in the art, as well as body fluids (e.g. blood or lymphatic fluid) from the recipient organism. A pharmacologically-acceptable carrier may additionally be selected from hydrogel materials that include, but are not limited to, non-fibrogenic alginate, agarose, alginic acid, carrageenan, collagen, gelatin, polyvinyl alcohol, poly(2-hydroxyethyl methacrylate), poly(N-vinyl-2-pyrrolidone) or gellan gum, either alone or in combination, or, alternatively, liquid, gelled, polymeric, co-polymeric or particulate formulations of aminated glucopolysachharides.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiment thereof and from the claims.